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Background Evidence supporting the effectiveness of therapeutic protocols for nonassociative immune‐mediated hemolytic anemia (na‐IMHA) is weak. Hypothesis/Objectives Investigate the efficacy of various drugs in na‐IMHA. Animals Two hundred forty‐two dogs. Methods Multi‐institutional retrospective study (2015‐2020). Immunosuppressive effectiveness was determined by time to packed cell volume (PCV) stabilization and duration of hospitalization through analysis by mixed model linear regression. Occurrence of disease relapse, death, and antithrombotic effectiveness, were analyzed using mixed model logistic regression. Results Use of corticosteroids vs a multi‐agent protocol had no effect on time to PCV stabilization (P = .55), duration of hospitalization (P = .13), or case fatality (P = .06). A higher rate of relapse (P = .04; odds ratio: 3.97; 95% confidence interval [CI]: 1.06‐14.8) was detected in dogs receiving corticosteroids (11.3%) during follow‐up (median: 28.5 days, range: 0‐1631 days) compared to multiple agents (3.1%) during follow up (median: 47.0 days, range: 0‐1992 days). When comparing drug protocols, there was no effect on time to PCV stabilization (P = .31), relapse (P = .44), or case fatality (P = .08). Duration of hospitalization was longer, by 1.8 days (95% CI: 0.39‐3.28 days), for the corticosteroid with mycophenolate mofetil group (P = .01) compared to corticosteroids alone. Use of clopidogrel vs multiple agents had no effect on development of thromboses (P ≥ .36). Conclusions and Clinical Importance Addition of a second immunosuppressive agent did not alter immediate outcome measures but might be associated with a reduction in relapse. Use of multiple antithrombotic agents did not reduce incidence of thrombosis.

Background Endogenous carboxyhemoglobin (COHb) production is a byproduct of hemoglobin metabolism. Hypothesis Blood carboxyhemoglobin concentrations are higher in cats with hemolytic anemia (HA). Animals Twenty cats with HA, 29 cats with non‐HA, and 22 controls were prospectively followed. Methods Blood tests were performed upon admission. The Mann–Whitney and Kruskal–Wallis tests were used for comparisons. Receiver‐operating characteristic (ROC) analyses tested COHb as a marker of HA or survival. Results The HA group included 17 cats with immune‐mediated HA and 3 with Heinz body (HB) anemia. In the non‐HA group, leading diagnoses included kidney disease (n = 14), acute/chronic blood loss (n = 11) and pancytopenia (n = 3). Carboxyhemoglobin concentrations (median [IQR]) significantly differed between cats with HA (5.55% [1.9]) and cats with non‐HA (1.9% [0.7]) or controls (1.9% [0.67]; p < 0.001 for both), but not between the last two groups (p = 0.6). Among 13 nonanemic stray cats with significant HB formation, the median (IQR) COHb concentration was 6.1% (1.2). The area under the ROC curve for COHb as a predictor of HA among all anemic cats was 0.996 (95% CI, 0.985–1), with an optimal cut‐off point of 2.95% yielding a sensitivity/specificity of 95% (95% CI, 76%–99%) and 100% (95% CI, 88%–100%), respectively. Survival and COHb concentrations were not associated in either group. Conclusions and Clinical Importance COHb proved a useful ancillary test in cats with suspected HA. Nevertheless, endogenous COHb production occurs with the absorption of large hematomas, not studied herein, or during hemolysis irrespective of anemia. These caveats must be considered when applying the present findings to the clinical and research setting.

Immune‐mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune‐mediated erythrocyte destruction, and adverse consequences of long‐term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence‐based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.

Background Red blood cells (RBC) are uniquely susceptible to oxidative injury. Oxidative stress is both a cause for, and effect, of anemia in people but this has been minimally documented in dogs. Objective To describe direct and indirect markers of oxidative stress in anemic dogs. Hypothesis Anemic dogs will have oxidative stress when compared to healthy dogs. Animals Forty‐seven dogs with anemia (10 with hemolytic anemia) and 70 healthy control dogs. Methods Prospective, cross‐sectional study. Anemic dogs were identified from the patient population, and medical records were reviewed to classify the anemia as hemolytic or nonhemolytic. Flow cytometry was used to detect reactive oxygen species (ROS) in erythrocyte isolates. Reduced glutathione (GSH) concentrations were measured in both plasma and hemolysate samples, and vitamin E was measured in serum. Results Anemic dogs (both hemolytic and nonhemolytic) had significantly lower median RBC hemolysate GSH concentrations (3.1 μM [0.4‐30.8]) when compared to healthy dogs (7.0 μM [0.5‐29.7]; P = .03). Dogs with hemolytic anemia had significantly higher median plasma GSH (7.6 μM [0.4‐17.8]) when compared to dogs with nonhemolytic anemia (1.6 μM [0.01‐7.1]; P = .04) and healthy dogs (2.8 μM [0.1‐29.9]; P < .0001). Reactive oxygen species were detectable in all samples, but there was no difference in ROS or vitamin E between groups. Conclusions and Clinical Importance Oxidative stress is present in anemic dogs. Derangements in biomarkers of oxidative stress are different in dogs with hemolytic anemia and nonhemolytic anemia.

Background Endogenous production of carbon monoxide during hemoglobin metabolism leads to the formation of carboxyhemoglobin. Carboxyhemoglobin concentration is abnormally high in humans with hemolytic anemia (HA). Hypothesis Measurement of carboxyhemoglobin concentration can discriminate HA from other forms of anemia. Animals Twenty‐seven dogs with HA (immune‐mediated HA, n = 22; microangiopathic HA, n = 5), 27 dogs with non‐HA (kidney disease, n = 14; immune‐mediated thrombocytopenia, [n = 6]; miscellaneous, n = 7) and 24 nonanemic control dogs. Methods Prospective cohort study. Carboxyhemoglobin quantification, a CBC and biochemistry profile were performed upon admission, and survival to hospital discharge and at 30 days were the measured outcomes. Groups were compared by the Mann‐Whitney and Kruskal‐Wallis tests. Receiver‐operator characteristic (ROC) analyses were used to examine the predictive utility of carboxyhemoglobin for the diagnosis of HA in anemic dogs. Results Carboxyhemoglobin (median [interquartile range]) differed between dogs with HA (7.7% [2.5%]) and non‐HA (3.6% [1.05]; P < .001) and dogs with HA and nonanemic dogs (3.5% [0.65%]; P < .001). No difference was detected between nonHA and nonanemic dogs. The area under the ROC curve for carboxyhemoglobin as predictor of HA in anemic dogs was 0.997 (95% CI, 0.99‐1.00). Three optimal cut‐off points were identified, including 5.05%, 4.55% and 4.85%, with corresponding sensitivity/specificity of 92.6%/100%, 100%/92.6% and 96.3%/96.3%, respectively. Neither carboxyhemoglobin nor any of the CBC or chemistry analytes were associated with survival. Conclusions and Clinical Importance Carboxyhemoglobin proved an excellent predictor of HA in dogs and might constitute a useful, ancillary tool for diagnosing and monitoring hemolytic anemias.

Immune‐mediated hemolytic anemia (IMHA) causes severe anemia in dogs and is associated with considerable morbidity and mortality. Treatment with various immunosuppressive and antithrombotic drugs has been described anecdotally and in previous studies, but little consensus exists among veterinarians as to the optimal regimen to employ and maintain after diagnosis of the disease. To address this inconsistency and provide evidence‐based guidelines for treatment of IMHA in dogs, we identified and extracted data from studies published in the veterinary literature. We developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria, explanation of treatment regimens, and validity of statistical methods. In combination with our clinical experience and comparable guidelines for humans afflicted with autoimmune hemolytic anemia, we used the conclusions of this process to make a set of clinical recommendations regarding treatment of IMHA in dogs, which we refined subsequently by conducting several iterations of Delphi review. Additionally, we considered emerging treatments for IMHA in dogs and highlighted areas deserving of future research. Comments were solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted for publication. The resulting document is intended to provide clinical guidelines for management of IMHA in dogs. These guidelines should be implemented pragmatically, with consideration of animal, owner, and veterinary factors that may vary among cases.

Background No tick‐borne pathogens (TBPs) causing haemolytic anaemia in cattle have been reported, except Theileria orientalis and complete blood count (CBC) profile is the only haematological parameter to determine the severity of regenerative haemolytic anaemia. Objectives To identify the causative agents of TBP‐induced haemolytic anaemia and determine haematological parameters that indicate haemolytic anaemia in grazing cattle. Methods Eighty‐two Korean indigenous cattle (Hanwoo) were divided into two groups: grazing (n = 67) and indoor (n = 15) groups. CBC and serum biochemistry were performed. PCR was conducted using whole blood‐extracted DNA to investigate the prevalence of TBPs. Results TBP‐induced haemolytic anaemia was observed in the grazing group. In grazing cattle, co‐infection (43.3%, 29/67) was most frequently detected, followed by T. orientalis (37.6%, 25/67) and Anaplasma phagocytophilum infections (1.5%, 1/67). In indoor cattle, only co‐infection (20%, 3/15) was identified. Grazing cattle exhibited regenerative haemolytic anaemia with marked monocytosis, mild neutropenia, and thrombocytopenia. According to grazing frequency, the 1st‐time grazing group had more severe anaemia than the 2nd‐time grazing group. Elevations in indirect bilirubin and L‐lactate due to haemolytic anaemia were identified, and correlations with the respective markers were determined in co‐infected grazing cattle. Conclusions Quantitative evaluation of haematocrit, mean corpuscular volume, and reticulocytes (markers of regenerative haemolytic anaemia in cattle) was performed for the first time. Our results show that, in addition to T. orientalis, A. phagocytophilum is strongly associated with anaemia. The correlation between haemolytic anaemia severity and haematological parameters (indirect bilirubin, reticulocytes, and L‐lactate) was confirmed. In grazing Korean indigenous cattle, haemolytic anaemia caused by Theileria orientalis and Anaplasma phagocytophilum has been observed. Reticulocytosis, a marker of regeneration, was quantitatively expressed with an increase in indirect bilirubin and a decrease in haptoglobin. In addition, indirect bilirubin, L‐lactate and reticulocytes were correlated with the severity of anaemia after HCT.

Mycoplasma ovis is a small, pleiotropic bacterium, which parasitizes the external surface of erythrocytes of several species of artiodactyl mammals, especially sheep and goats. We here report an outbreak of ovine mycoplasmosis in a sheep flock of a private ranch (Universidad Veracruzana) in Veracruz, Mexico. For the identification of Mycoplasma and other hemoparasitic bacterial agents, we stained blood smears with the DiffQuick® technique and additionally amplified several fragments of 16S rDNA gene. We detected the presence of morulas in erythrocytes from 30 sick female adult sheep, and found Mycoplasma ovis DNA in all of them. Furthermore, three of these animals also tested positive for Anaplasma ovis. Our findings represent the first record of M. ovis and A. ovis in an outbreak of hemolytic anemia in a sheep flock, leading to severe livestock loss in a ranch of Mexico. This study highlights the importance of establishing an active surveillance of both pathogens in the country.

Immune-mediated hematologic diseases (IMHD) including immune-mediated hemolytic anemia (IMHA) and immune thrombocytopenia (ITP) can cause severe disease in dogs. The underlying immune system abnormalities associated with these conditions is not known. The hypotheses of this study were that dogs with IMHD would have increased frequencies of CD4+ and CD8 + lymphocytes, decreased frequencies and numbers of T regulatory cells, and increased frequencies of interleukin (IL)-17 + lymphocytes. Fifteen dogs with newly diagnosed IMHA or ITP and 15 healthy control dogs were recruited for this prospective study. Flow cytometry was used to enumerate CD4 + lymphocytes, CD8 + lymphocytes, T regulatory (CD4 + CD25 + Foxp3+) cells, and lymphocytes secreting IL-17 in dogs with IMHD at diagnosis, then 2 and 4 days after starting immunosuppressive treatment. Median proportion of CD4+ (Day 0: 3.4%, Day 2: 3.3%) and CD8+ (Day 0: 1%, Day 2: 0.6%) cells was lower in dogs with IMHD compared to control dogs (CD4 + 22.8%, CD8 + : 13.6%; P < 0.0001 for each). Additionally, T regulatory cells were reduced in IMHD dogs at Day 0 (0.2% versus 0.6% of total lymphocytes, P = 0.0025). Dogs with IMHD had a higher proportion of lymphocytes positive for IL-17 at Day 2 (1.3%) compared to control dogs (0.4%, P = 0.0024). Dogs with IMHD have immune system alterations at diagnosis and during early treatment characterized by a deficiency in T regulatory cells and an increase in IL-17 + lymphocyte. These changes might contribute to the pathogenesis of IMHA and ITP.

Background Although precursor‐targeted immune‐mediated anemia (PIMA) is thought to be caused by immune targeting of erythroid precursors (nucleated RBCs, nRBCs), its pathogenesis is unknown. Immunoglobulin G (IgG) or phosphatidylserine (PS) may promote nRBC destruction in PIMA. Hypothesis Dogs with PIMA have increased nRBC IgG and PS, and dogs with immune‐mediated hemolytic anemia (IMHA) have increased RBC PS compared to healthy dogs. Animals Blood from 20 healthy dogs and from dogs with IMHA (11) or other (non‐IMHA) conditions (9), and marrow aspirates with or without blood from 10 healthy dogs and from dogs with PIMA (17) or other (non‐IMHA, non‐PIMA) conditions (7). Methods Marrow nRBC stages were separated by density gradient. Flow cytometry was used to assess the percentage of RBCs or nRBCs with increased IgG or PS. Results Red blood cell (RBC) IgG positivity was increased in 9/11 IMHA dogs and 0/9 non‐IMHA dogs. Red blood cell PS positivity was increased in 10/11 IMHA dogs and 2/9 non‐IMHA dogs. Five of 17 PIMA dogs had increased nRBC IgG positivity in mid‐ or late‐stage fractions, whereas all 7 non‐PIMA dogs were negative. Mid‐ and late‐stage erythroid precursor PS was significantly higher in PIMA dogs compared to healthy dogs. Five of 14 PIMA dogs had increased RBC IgG positivity. Conclusions Immunoglobulin G and PS may promote destruction of nRBCs in PIMA dogs; PS may promote destruction of RBCs in IMHA dogs.